The Murine Anti-Human Common γ Chain Monoclonal Antibody CP.B8 Blocks the Second Step in the Formation of the Intermediate Affinity IL-2 Receptor

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Abstract
A murine monoclonal antibody, CP.B8, specific for the extracellular portion of the human common γ (γc) chain, and its Fab fragment are shown to block the binding of IL-2 to COS-7 cells transfected with the cDNA for the full-length IL-2 receptor β (IL-2Rβ) and γc chains, components which together comprise the intermediate affinity IL-2 receptor (IL-2R) expressed on the surface of resting T cells, NK cells, and on certain intestinal epithelial cells. To investigate the mechanism of this inhibition, the extracellular portions of the IL-2Rβ and γc chains were expressed and purified, and their interactions with each other and with IL-2 were studied by gel filtration and by surface plasmon resonance (SPR). By gel filtration, a stable ternary complex was formed by association of the three proteins, while no stable binary complexes were detected between any two of the three proteins. By SPR analysis, IL-2 was shown to associate rapidly with IL-2Rβ, forming a binary complex with an equilibrium dissociation constant (Kd) of 800 nM, which permitted subsequent association of the γc chain. Dissociation of the IL-2/IL-2Rβ/γc chain complex was significantly slower than dissociation of the IL-2/IL-2Rβ complex. Using these model systems, we tested the ability of mAb CP.B8 to inhibit the association of the γc chain with IL-2 and IL-2Rβ. By gel filtration, mAb CP.B8 formed a stable complex with the γc chain, preventing its association with IL-2 and IL-2Rβ. MAb CP.B8 was also capable of dissociating the γc chain already complexed with IL-2 and IL-2Rβ. SPR analysis confirmed these findings and showed, in addition, that the Fab fragment of CP.B8 was also capable of inhibiting the association of the γc chain with the IL-2/IL-2Rβ complex. We conclude that mAb CP.B8 blocks the second step in the formation of the intermediate affinity IL-2R on the surface of transfected COS-7 cells by binding at or close to a region on the γc chain that is involved in contact with IL-2 and/or IL-2Rβ.

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